The persistence of pathogenic bacteria and the processes that enable them to infect their hosts require the integration of multiple signals. Understanding how bacteria orchestrate multiple processes is critical for our ability to design strategies to inhibit their persistence and proliferation in the environment, including pathogens during host infection, and will result in the improvement of public health. The localization of proteins and organelles at the cell poles is an important component of bacterial interaction with the environment;many pathogenic bacteria localize virulence factors to their cell poles. The long-term goal of this research is to use the model organism Caulobacter crescentus to understand how cell division, polar localization, morphological changes, and cell physiology are coordinated during bacterial growth. This bacterium synthesizes a number of polar organelles in a specific order during its asymmetric life cycle, in which a motile swarmer cell differentiates into a sessile stalked cell that then divides to produce a swarmer and a stalked cell. The synthesis of polar organelles is regulated by an elaborate signal transduction network, in which multiple inputs are integrated. These inputs include checkpoints that coordinate organelle synthesis with cell division and physiology. Polar development culminates in the morphogenesis of a thin cell envelope extension at the pole, known as the stalk, which improves nutrient uptake. The main objective of this research is to identify the regulatory mechanisms that couple polar development to cell division, and to determine how this regulatory pathway culminates in stalk synthesis. Inhibition of cell division activates a checkpoint that halts development of the new cell pole after flagellum synthesis: the pili, the holdfast, and the stalk are not synthesized. The first goal of this research is to determine the function of PodJ, a developmental regulator, in the cell division checkpoint. A full length form of PodJ, PodJL, localizes to the pole opposite the stalk in early predivisional cells, and is processed into a shorter form, PodJs, at the time of cell division. PodJL processing is inhibited when cell division is blocked. All known mutants that allow polar development when cell division is inhibited restore PodJL processing, suggesting that the processing of PodJL provides a molecular switch that signals the progression of specific developmental stages. The mechanisms and regulation of PodJL processing and their role in polar development during normal growth will be determined. The second goal is to determine the role of a response regulator, TacA, required for the inhibition of polar development and PodJL processing when cell division is inhibited. The mechanisms that regulate the TacA phosphorelay and how this pathway regulates PodJL processing will be determined. The third goal is to study genes required for stalk synthesis and regulation, including genes regulated by TacA, and to perform a detailed ultrasctucture analysis of stalk synthesis in wild-type and mutant strains using electron cryotomography. Our study will provide insight into how we can interfere with and inhibit the execution of the multiple processes required for the infection of pathogenic bacteria. PUBLIC HEALTH RELEVANCE: Pathogenic bacteria rely on the integration of multiple external and internal signals to respond to their environment and achieve effective host infection. This proposal uses the model bacterium Caulobacter crescentus to determine how bacteria integrate multiple regulatory pathways to produce specific advantageous morphologies. Understanding how bacteria orchestrate multiple processes is critical for our ability to inhibit their persistence, proliferation, and host infection, and will result in the improvement of public health.